Gut 1993; 34:279-283

CASE REPORT

Familial visceral myopathy associated with a

mitochondrial myopathy

R Lowsky, G Davidson, S Wolman, K N Jeejeebhoy, R A Hegele

AbstractA 27 year old man with intestinal pseudo-obstruction who developed parenteral nutritioninduced hyperlipidaemia and who also hadophthalmoplegia and an undifferentiatedmyopathy is described. Histological examina-tion of biopsy specimens and molecularanalysis show that this patient had both familialvisceral myopathy and a mitochondrialmyopathy, suggesting that a mitochondrialDNA mutation is the molecular lesion infamilial visceral myopathy.(Gut 1993; 34: 279-283)

Departments of Medicineand Pathology,University of Toronto,Toronto, Ontario,CanadaR LowskyG DavidsonS WolmanK N JeejeebhoyR A HegeleCorrespondence to:Dr R A Hegele, 30 BondStreet, Toronto, Ontario,Canada M5B 1W8.

Accepted for publication27 July 1992

The familial visceral myopathy syndromes are a

clinically heterogeneous group of rare disordersof unknown molecular origin. Segregationpatterns consistent with both autosomaldominant and autosomal recessive inheritancehave been described in affected kindreds.' Thehallmark of the syndromes is the histologicalintestinal abnormality characterised by smoothmuscle cell vacuolar degeneration, loss, andfibrosis, especially of the outer longitudinalmuscle layer.2The mitochondrial myopathies encompass a

distinct group of well described neurologicalsyndromes (Kearns-Sayre, myoclonic epilepsylactic acidosis and stroke-like syndromes(MELAS), myoclonic epilepsy and ragged redfibres (MERRF), and Leber's hereditary opticneuropathy) characterised histologically by thepresence of coarse subsarcolemmal red stainingaggregates in skeletal muscle, stained accordingto the modified Gomori trichrome method.3Expression of these syndromes frequentlyreflects impaired mitochondrial functionconsequent to mitochondrial DNA (mtDNA)changes,4 most commonly deletions within theregion spanning nucleotide bases 8000-9500.1Because mitochondria are acquired from the ova,mitochondrial myopathies are generallymaternally transmitted.6

Individuals affected by either familial visceralmyopathy7 or a mitochondrial myopathyt 9

manifest varying degrees of intestinal pseudo-obstruction, polyneuropathy, leukoencephalo-pathy, lactic acidosis, and ophthalmoplegia. Thenearly identical clinical features have promptedprevious suggestions of an association betweenthese two disorders.1'12 We describe a 27 year oldman with an undifferentiated myopathy. Histo-logical examination of biopsy specimens andmolecular analysis show that this patient had

both familial visceral myopathy and a mito-chondrial myopathy, suggesting that a mtDNAlesion is associated with familial visceralmyopathy.

Methods

PROBANDA 27 year old, non-smoking, heterosexualYugoslavian man presented with perforatedduodenal diverticula. His past medical historyincluded 10 years of diarrhoea alternating withconstipation, intermittent abdominal cramps,inability to maintain weight, and a recent onsetof muscle weakness with paraesthesia in alllimbs. He also complained of difficulty withconcentration. He was using no medications. Hisyounger brother had nearly identical complaintsand two sisters had died in infancy from unknowncauses (Fig 1). Physical examination showed thatthe patient had a profound loss of body fat andmuscle mass. His height, weight, and body massindex were 178 cm, 48 kg, and 15 1 kg/m2,respectively. Abdominal examination showed anacute abdomen without organomegaly; stoolswere positive for occult blood. Neurologicalexamination showed deficits in attention andconcentration. There was moderate ophthal-moparesis on upward gaze and mild bilateralweakness in flexion and extension of musclegroups in the legs. Proprioception, vibrationsense, and light touch were impaired in the legs.Cerebellar function and gait were normal.The patient had a partial duodenal resection

and a primary reanastomosis. He received totalparenteral nutrition (TPN) postoperatively.

PATHOLOGY STUDIESThe duodenal tissue obtained at surgery wasfixed and stained with haematoxylin and eosin. Agracilis muscle biopsy was examined withparaffin histology, a Gomori trichrome stain,and by electron microscopy.

MOLECULAR STUDIESTotal cellular DNA was extracted from 500 mgof frozen skeletal muscle using an establishedprocedure.'3 Mitochondrial DNA spanning theregion of 8530-9110 nucleotides was amplifiedusing the Taq polymerase chain reaction (PCR).Total cellular DNA (10 [tg) was incubatedin a Tempcycler (Coy) with Taq polymerase

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Family pedigree

33 31 29

hFigure 1: Pedigree structure showingfirst degree relatives. Closed symbols represent affected subjects, hatched symbols clinicallysuspect subjects, and open symbols unaffected subjects. The proband is depicted by the arrow.

(BRL) and the oligonucleotide primers5'-ACGAAAATCTGTTCGCTTCA-3' (span-ning mtDNA nucleotides 8530-8550) and5'-ATTGTGAAGATGATAAGTGT-3' (span-ning mtDNA nucleotides 9090-9110). '" Thetarget domain was amplified in 30 three-stepcycles: denaturing at 94°C for 90 seconds,annealing at 55°C for 90 seconds, and extensionat 72°C for 240 seconds. The amplified productwas then used as a probe for Southern blotanalysis and as a template for nucleotide sequenceanalysis. Total cellular DNA was digested withendonuclease BamHI, EcoRI, or HindIII.Southern analysis was performed as described"3using the PCR amplified mtDNA productlabelled with the PhotoGene Nucleic AcidDetection System (BRL) as a probe. The PCRamplified mtDNA product from the probandand a control were sequenced directly using thePCR oligonucleotide primers in dideoxysequencing.

Results

CLINICAL FEATURESThe patient's abnormalities are given in theTable. The most important were intestinalpseudo-obstruction, polyneuropathy, leuko-

Clinical abnormalities found in a patient with familial visceral myopathy and a mitochondnialmyopathy

Laboratory test Finding Interpretation

Barium studies:Upper gastrointestinal Normal oesophagus, mild dilatation of stomach, Intestinal pseudo-

multiple diverticula within small bowel with obstructionstasis of contrast material

Lower gastrointestinal Few colonic diverticula with dilatationNerve conduction Marked delay/absence of nerve conduction in Chronic

studies upper and lower limbs demyelinatingneuropathy

Magnetic resonance Diffuse white matter lesions Leukoencephalopathyimaging of cranium

Serum lactate >3 5 mmol/l Chronic lactic acidosisFasting RQ >1 Incompetent fat/lipid

metabolismTotal parenteral Milky serum, cholesterol > 18 mmol/l, Total parenteral

nutrition feeding triglycerides > 15 mmolUl, no weight gain after nutrition induced3 months hyperlipidaemia

encephalopathy, and many metabolic abnormali-ties that included a chronic lactic acidosis, totalparenteral nutrition induced hyperlipidaemia,and a fasting respiratory quotient exceeding 1 0.The serum concentration of carnitine and thecarnitine palymitol transferase activity werenormal. A normal glucose response to fastingwas observed (data not shown). Investigations ofthe proband's brother were similar, with theexceptions of the absence of total parenteralnutrition induced metabolic disturbances andthe presence of type II diabetes mellitus.

PATHOLOGYThe surgical duodenal specimen from theproband showed mild villous blunting and aslightly increased cellularity of the laminapropria. There was smooth muscle cell loss withfibrosis, especially within the outer longitudinalmuscle layer (Fig 2A). The myenteric plexus wasunremarkable, containing numerous and normalganglion cells. Electron microscopy examinationconfirmed myocyte loss and the presence ofcollagen fibrosis (Fig 2B). In clinical terms, thesehistological findings established a diagnosis offamilial visceral myopathy.The gracilis muscle biopsy showed scattered

degenerating and regenerating fibres with noinflammation, fibrosis, or neurogenic changes.The Gomori trichrome stained sections showedsubsarcolemmal aggregates of red granularmaterial corresponding to abnormal mito-chondria (not shown). Electron microscopyshowed aggregates of mitochondria with intra-mitochondrial inclusions consisting of closelyapposed parallel membranes resembling 'zippers'or 'parking lots' (Fig 3). These skeletal musclefibre changes were considered pathognomonic ofa mitochondrial myopathy.

MOLECULAR ANALYSESSouthern blot analysis of BamHI, EcoRI, andHindlIl digested muscle DNA from our patient,hybridised with a labelled mtDNA probe,

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Familial visceral myopathy associated with a mitochondrial myopathy

showed fragments of predicted size whencompared with a normal control (Fig 4). Noheterogeneity ofmtDNA species (heteroplasmy)was found.The nucleotide sequence of the mtDNA

domain 8530-9110 nucleotides (a region thatencodes subunit 6 of ATPase) from the probandwas identical to the wild type sequence, exceptfor a single base substitution of a thymidine forcytosine at position 8634 (Fig 5). This substi-tution was silent at the protein level: a tyrosineresidue was translated both from the patient andthe wild type mtDNA.

DiscussionWe present a patient with the concomitantdiagnoses of familial visceral myopathy and amitochondrial myopathy. The diagnosis offamilial visceral myopathy was based on charac-teristic clinical features and histological abnor-malities within the small bowel. The diagnosis ofa mitochondrial myopathy was based on thepathognomonic findings within the mitochondriaof skeletal muscle cells when examined byGomori trichrome and electron microscopy. Thefindings in this patient suggest that familialvisceral myopathy and mitochondrial myopathiesmay have a similar molecular origin and couldrepresent two points on the spectrum of a singledisease.

Familial visceral myopathy and the mito-chondrial myopathies have been distinguishedfrom each other on the basis of muscle typeaffected. Familial visceral myopathy has beenassociated with smooth muscle cell abnormali-ties, whereas the mitochondrial myopathies havebeen characterised by skeletal muscle cellabnormalities. None of the reported cases offamilial visceral myopathy, however, haveundergone a Gomori trichrome stained musclebiopsy.' 27 5-22 Likewise, none of the reportedcases of mitochondrial myopathies, specificallythose in which patients were predominantlyaffected with a chronic intestinal pseudo-obstruction, have reported the histologicalfindings of the small bowel wall.9 It is thuspossible that the failure to detect coexistentabnormalities in these conditions is the result of alack of specific histological examination.There are similarities in the expression of the

clinical phenotypes of familial visceral myopathyand of the mitochondrial myopathies. Forexample, intestinal diverticulosis is a charac-teristic accompaniment of familial visceralmyopathy and has also been described in themitochondrial myopathies.9 Furthermore, extra-intestinal neurological manifestations in familialvisceral myopathy include ophthalmoplegia,megacystis, dementia, and seizure dis-orders,2 192022 features that are also characteristicof mitochondrial disorders. In addition, apatient with familial visceral myopathy had lactic

Figure 2: (A) Lightmicroscopy ofduodenumshowing myocyte loss andfibrous replacement (arrow)within the outer longitudinalmuscle layer. (B) Electronmicroscopy ofsame specimenshowing myoctye loss andfibrous replacement withcollagen (upper halfoffigure).

Figure 3: Electron microscopy ofgracilis muscle biopsy tissue showing an aggregate ofmitochondria containing 'parking lot' inclusions.

acidosis, total parenteral nutrition inducedhyperlipidaemia, right bundle branch block,polyneuropathy, and leukoencephalopathy,7features that are characteristic of mitochondrialdisorders. Our patient had many of these clinicalfeatures.A factor that could theoretically distinguish

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I 2 34 5

16'65107*4 kb

b55kb

LEGEND:probe mtD A(853091 10)I-anea- I- = MFV subject BamHilane 2-- normallane3e =RM3subect EcoRllane 4= normal' Hindilllane: 5-= FVM subjct HindNilFigure 4: Southern analysis ofmuscle mitochondrial (mt) DNA from the proband and controlsubject. The band sizes are shown. Lane I represents mtDNA from the proband after digestionwith BamHI. Lanes 2 and 3 compare mtDNA from a normal subject with mtDNA from theproband, respectively, after digestion with EcoRI. Lanes 4 and 5 compare mtDNA from thenormal subject with mtDNA from the proband, respectively, after HindIII enzymaticdigestion. Fragments ofexpected size were observed in each case: digestion with BamHIproduced a 16 5 kbfragment corresponding to the linearised, wild type mtDNA. Digestion withEcoRI and HindIII produced the expectedfragments of 7-3 and 5 5 kb, respectively.

A C O T

1'5,

between familial visceral myopathy and themitochondrial myopathies is their mode ofinheritance. Familial visceral myopathy isinherited as an autosomal trait,' whereas themitochondrial myopathies classically display amaternal inheritance. Our patient had anaffected male sibling and two female siblingswho had both died in infancy, suggesting auto-somal dominant inheritance with variablepenetrance. This would be compatible withfamilial visceral myopathy but not with theclassical mitochondrial myopathies. However,three pedigrees with mitochondrial myopathieshave been reported in which defective paternalgenes affecting nuclear control over mtDNAreplication resulted in large scale mtDNAdeletions.'4 Another study has shown paternaltransmission of mtDNA from males in back-crossed mice.2 Thus, the mode of transmissiondoes not necessarily distinguish familial visceralmyopathy from a mitochondrial myopathy.The genetic defect in familial visceral

myopathy is unknown. In contrast, 40% ofpatients with mitochondrial myopathies havelarge scale deletions in muscle mtDNA usuallyincluding the region between nucleotides 8000and 9500. In addition, one form ofMERRF andone form of Leber's hereditary optic neuropathyare caused by point mutations within the mito-chondrial genome.'6 Our patient had no grossstructural changes of the muscle mtDNAgenome. Furthermore, detailed sequenceanalysis ofour patient's muscle mtDNA betweennucleotides 8530 and 9110, a region that is oftenaffected in mitochondrial myopathies, showedonly a silent DNA polymorphism. The moleculardefect in our patient remains to be determined. Itis still possible that a single lesion affecting themtDNA genome is responsible. The finding of

mtDNAA COGT

C -

Figure 5: Direct dideoxysequencing ofmusclemitochondrial (mt) DATA ofthe proband and a controlsubject. The readingframeis 5' to 3' from bottom to topand the mtDNA sequencesare compared for a normalcontrol and the proband. Asingle base substitution ofthymine (T) for cytosine (C)at position 8634 was found.The codons each specifytzyosine.

l --~~~~~T-34l 833

AMA TAT Cit

AMA TACCi

Tyr

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Familial visceral myopathy associated with a mitochondrial myopathy 283

concomitant familial visceral myopathy andmitochondrial myopathy in this family suggestthat familial visceral myopathy may be a mito-chondrial myopathy.

We acknowledge the assistance of Drs G Gardiner, C Bergeron,K Siminovitch, J Abrahamson, and L Tu. This work wassupported by a grant from the Hospital for Sick Children ResearchFoundation.

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